Engagement of the receptor protein Fas/TNFRSF6 by its ligand, FasL/TNFSF6, triggers apoptotic cells death via well-understood mechanisms involving the assembly of a death-inducing signaling complex (DISC). Fas can also trigger a potent pro-inflammatory program of gene expression in some epithelial cells, such as keratinocytes. The mechanisms of the inflammatory responses to Fas ligation are not understood despite the fact that Fas and FasL have been implicated in inflammatory conditions, from eczematous dermatitis to neuroinflammation in Parkinson's disease. Experimental evidence presented in the Preliminary Studies and our published research suggests that the pro-inflammatory actions of Fas are mediated by a Fas 'signalosome'for which we propose the name 'Fas-activated Inflammation-Signaling Complex'(FISC). In keratinocytes, FISC triggers inflammatory gene expression via signal-transduction pathways that activate the nuclear factor-kappa B (NF-kB), the stress-activated protein kinases (SAPK) JNK and p38 MAP kinase, and the epidermal growth factor receptor (EGFR)-Ras-ERK MAP kinase cascade. Initial biochemical and functional characterization of FISC demonstrates the essential roles of the adaptor protein FADD and the initiator caspase 8 (CASP8) which are also participants in the DISC. However, unlike DISC, FISC does not require the protease activity of CASP8 for its function. The adaptor protein TRADD and the kinase RIP1 were also identified as mediators of the pro-inflammatory responses to Fas engagement. Downstream of FISC, Fas ligation causes the activation of the TGFb-Activated Kinase-1 (TAK1) and the Inhibitor of NF-kB-a (IkBa)-phosphorylating Kinase-b (IKKb). Research in this proposal will characterize, biochemically and functionally, the protein composition of FISC in human epidermal keratinocytes. We will identify downstream effectors of FISC and elucidate the mechanisms of regulation of FISC by FLIP proteins. Finally, we will reveal the mechanisms of crosstalk between the Fas and EGFR pathways. Elucidating the mechanisms of Fas-induced inflammation will improve our understanding of the involvement of Fas in pathology (from eczema to neuroinflammation) and treatment (such as immunotherapy of cancer). PUBLIC HEALTH RELEVANCE: This proposal aims at elucidating the mechanisms of non-apoptotic pro-inflammatory signaling through Fas ligand (FasL/TNSF6) and its receptor Fas/TNFRSF6.